Pharmaceutical composition comprising a combination of at least one immunotoxin and at least one mannose-containing polymer

ABSTRACT

The invention relates to pharmaceutical compositions comprising a combination of at least one immunotoxin and at least one mannose-containing polymer. This combination makes it possible to inhibit the rapid elimination of immunotoxins from the plasma after injection.

In French Pat. No. 78/27838 and the patent for Addition attached theretoNo. 79/24665 of the prior art, and in French patent Applications No.81/07596 and No. 81/21836, the Applicant Company described thepreparation of anticancer products, called conjugates, obtained by thecoupling, by means of a covalent bond, of the A chain of ricin withantibodies or antibody fragments directed against an antigen carried bythe cell to be destroyed. Products of this type are denoted in thepresent Application by the generic name of immunotoxins. The immunotoxinwhich is used in the present invention can be obtained from a natural,semisynthetic or synthetic toxin, toxic sub-unit or fragment of toxicsub-unit comprising polysaccharide groups containing mannose residues,in particular in the terminal position, irrespective of the constituentantibody and irrespective of the type of bond chosen to join theantibody of the toxin, toxic sub-unit or toxic fragment.

In its French Patent Applications No. 81/21836, No. 82/02091, No.82/04119, No. 82/04047 and No. 82/04547, the Applicant Company alsoshowed the ability of certain substances (ammonium salts, monovalentcarboxylic ionophores, methylamine, chloroquine and enzyme conjugatescapable of releasing ammonia) to potentiate the cytotoxic action ofimmunotoxins.

However, the therapeutic effects of activated or non-activatedimmunotoxins can only manifest themselves fully if the immunotoxin iscapable, with its antibody part, of becoming localized in vivo, in theactive form, on the target cell to be destroyed (a indispensablecondition for all expression of activity by immunotoxins). The abilityof the immunotoxin to become localized on the target depends first andforemost on the ability of the immunotoxin to remain in the bloodstreamand the extracellular fluids, in the active form, for sufficient periodsof time to reach its target cell and at sufficiently high concentrationsto give a high degree of occupation of the corresponding antigen sites.

The Applicant Company has carried out a large number of studies whichhave made it possible to establish the plasma elimination kinetics ofimmunotoxins after intravenous injection into various animal models. Ithas been found that, after injection, the plasma level of biologicallyactive immunotoxin decreases very rapidly and very substantially. Thus,in a typical case involving rabbits, in a model using an immunotoxinbuilt up by coupling the A chain of ricin, by means of a link containinga disulfide bridge, with a monoclonal antibody directed against theantigen T65 of human T lymphocytes, it is found that 97% of theimmunotoxin present in the bloodstream at time 0 after injectiondisappears in 30 minutes and 99.9% disappears in 17 hours. The resultsobtained are analogous if the link joining the antibody to the A chainof ricin contains a thioether bond instead of a disulfide bond. Thisrapid disappearance of the immunotoxin quite obviously detracts from theexpression of its complete cytotoxic capacity, the immunotoxin beingprevented from durably saturating a high proportion of the targetantigens carried by the cells to be destroyed. Moreover, a comparison ofthe plasma elimination kinetics of immunotoxins with those of thecorresponding unconjugated antibodies shows by contrast that--as is wellknown--the antibodies remain in the plasma at a high level forrelatively long periods of time. Now, even in the most highly purifiedimmunotoxin preparations, there is always a certain residual level ofunconjugated antibodies. Due to the effect of the differential rates ofelimination of immunotoxins and antibodies, the unconjugated antibodies,which are initially very much in the minority, gradually become themajority component after a few hours, so these antibodies graduallycompete to become powerful antagonists for the fixation of theimmunotoxins to their targets.

These studies clearly show the value of enhancing the persistence ofimmunotoxins in the plasma, in their active form, so as to increase boththe duration and degree of occupation of the target antigens andconsequently to improve the therapeutic effects of the immunotoxins.

The present invention relates to a pharmaceutical combination whichmakes it possible to inhibit the rapid elimination of immunotoxins fromthe plasma after injection, without adversely affecting thecharacteristic intrinsic properties of the immunotoxins. It has beenfound that the presence in the plasma of a combination of at least oneimmunotoxin and at least one mannose-containing polymer inhibits therapid elimination of immunotoxins from the plasma after introduction ofthe immunotoxin in the plasma without adversely affecting thecharacteristic intrinsic properties of the immunotoxins.

Surprisingly, it has been found, according to the present invention,that mannanes constitute a particularly valuable type of substance forincreasing the plasma levels of immunotoxins. The term mannane is usedhere to denote any polyoside or polysaccharide carbohydrate polymerwhich has an average molecular weight greater than 1000 and contains alarge proportion of mannose residues, more particularly from 20 to 100%of mannose residues, irrespective of the type of osidic linkage joiningthese mannose residues to one another or to other sugars. In particular,and by way of a nonlimiting example, it is possible within the terms ofthe present invention to use natural mannanes isolated from yeasts (forexample Saccharomyces cerevisiae), i.e. the carbohydrate fraction of apeptidoglycan belonging to the cell wall of these yeasts. Theprotein-mannane complex is a mixture of macromolecules in which thepolysaccharide component represents 50 to 90% of the complex. Themannane fraction is itself a polymer of D-mannose. It consists of aframework of mannose residues coupled in the α1→6 position, with addedside-chains of different lengths containing α1→3 and α1→2 bonds.

Surprisingly, mannane, used at doses in which it shows no toxicity tothe animal, either on its own or in association with the immunotoxin,makes it possible to increase the plasma concentration of immunotoxinsby an extremely large factor (of the order of 100) for prolongedperiods, thereby considerably improving their localization on the targetand avoiding fixation inhibition phenomena due to the presence of freeantibodies in the preparations, as indicated previously.

The noteworthy absence of toxicity of mannanes makes them preferredsubstances for pharmaceutical use in association with immunotoxins.Association of the immunotoxin with the mannane does not significantlyincrease the inherent toxicity of the immunotoxin, nor does thisassociation interfere with the specific cytotoxicity propertiescharacteristic of the immunotoxins in the presence or absence of thepotentiators already described.

Furthermore, experiments involving in vivo localization of theradiolabeled immunotoxin injected into animals with no specific targethave shown that the conjugate becomes localized preferentially in theliver during the first few minutes after injection. The same applies tothe A chain, which follows the same pattern when injected in theuncoupled form. This strongly suggests that the immunotoxin becomesfixed in the liver via the A chain of ricin contained in theimmunotoxin. It is known that the A chain of ricin is a glycoproteinwhose polyosidic groups comprise mannose residues andN-acetylglucosamine residues, the mannose residues being in the terminalposition (Agri. Biol. Chem. (1978) 42, 501). Also, receptors capable ofrecognizing glycoproteins containing these terminal mannose residueshave been found to exist in the liver.

It has thus been shown that the glycoproteins recognized by thesereceptors--the latter being present essentially on the Kupffercells--are rapidly eliminated from the bloodstream by fixation to thesecells, which metabolize them. This is well documented especially in thecase of β-glucuronidase and in the case of ribonuclease B (Arch.Biochem. Biophys. (1978) 188, 418; Advances in Enzymology, edited by A.Meister, New York (1974); Pediat. Res. (1977) 11, 816).

Taken as a whole, this information shows that the rapid elimination ofimmunotoxins can be explained by the recognition of the mannose residuesof the A chain of ricin by the hepatic cells, in particular the Kupffercells. The property of mannanes to inhibit the rapid plasma eliminationof immunotoxins containing the A chain of ricin is also easily explainedby the fact that the mannanes administered occupy the receptor cells ofthe glycoproteins and therefore oppose the recognition, by thesereceptors, of the polyosidic units carried by the A chain of ricin or byany conjugate containing the latter.

The property of mannanes to inhibit the rapid plasma elimination ofimmunotoxins containing the A chain of ricin is equally applicable, forthe reasons indicated above, to the uncoupled A chain of ricin or to anynatural, semi-synthetic or synthetic hybrid molecule containing the Achain of ricin, and in particular to any immunotoxins containing the Achain or ricin, irrespective of the type of bond chosen to join theantibody to the A chain of ricin. For the same reasons, this property ofmannanes is more generally applicable to any immunotoxin, whatever thetoxin used to produce it, provided this toxin or toxic sub-unitcomprises polysaccharide groups containing mannose residues, inparticular in the terminal position, regardless of the constituentantibody and regardless of the type of bond chosen to join the antibodyto the toxin or toxic sub-unit.

The examples which follow provide a better understanding of theinvention without limiting its scope.

EXAMPLE 1

The purpose of this example is to demonstrate the changes in eliminationkinetics of immunotoxins (as well as their constituent components) inthe presence or absence of mannane.

A--The following procedures were used:

(a) Measurement of the elimination kinetics of the immunotoxin IT-T101

The conjugate called IT-T101 is obtained by reacting an antibodydirected against human T cells (antibody T101 directed against theantigen T65), substituted by an activated disulfide group, with the Achain of ricin. The preparation and cytotoxic properties of thisconjugate were described in French Patent Application No. 8121836 in thename of the Applicant Company. The conjugate IT-T101 is administered torabbits by a single injection into a vein in the ear. The quantityinjected corresponds to 1.25 mg of immunotoxin per kg of body weight,i.e. 0.415 mg/kg expressed as A chain and 0.835 mg/kg expressed asantibody. Blood samples are taken at intervals on heparin. The plasma isanalyzed by means of a radioimmunometric assay denoted hereafter by theabbreviation RIM-1.

This technique has the advantage of determining the immunotoxin withoutmodifying it. This determination is carried out in microtitration plates(for example: "NUNC-TSP screening system", Poly Labo Block, France), thecover of which is provided with hyperabsorbent spikes dipping into thewells in the base. These spikes constitute the solid phases. Sheepantibodies directed against the A chain of ricin (denoted hereafter bythe abbreviation Acl), purified by affinity chromatography, are adsorbedonto the solid phases. To do this, 200 μl of a solution of Aclcontaining 10 μg/ml in a buffer which is 20 mM in respect of phosphate,pH 7, and 150 mM in respect of NaCl are divided up into the wells. Thespikes are brought into contact first with the solution of Acl for 24hours at 4° C. and then with fetal calf serum for 3 hours at 20° C. inorder to saturate all the fixation sites. The saturated immunoabsorbentis then brought into contact for 3 hours at 20° C. with the plasmasamples at different dilutions, or with solutions of immunotoxin IT-T101of known concentrations in order to establish the calibration curve.Washing is carried out with a buffer which is 20 mM in respect ofphosphate, pH 7, and 150 mM in respect of NaCl, and the immunoabsorbentis then brought into contact for 2 hours at 20° C. with goat antibodiesdirected against mouse IgG, which have been purified by affinitychromatography and radiolabeled (denoted hereafter by the abbreviationAc2). The Ac2 is radiolabeled with iodine 125 in the presence ofchloramine T by the method of Greenwood and Hunter (Biochem. J., (1963)89, 114); the specific activity of the radiolabeled Ac2 is 5 to 10μCi/mg. 10⁶ cpm of radiolabeled Ac2 are introduced, in a volume of 200ml, into a buffer which is 20 mM in respect of phosphate, pH 7, and 150mM in respect of NaCl and contains 0.1% of bovine serum albumin. Afterwashing in a buffer which is 20 mM in respect of phosphate, pH 7, and150 mM in respect of NaCl, the spikes are detached and the quantity ofbound Ac2 is measured by counting the radioactivity. The immunotoxinconcentration in the samples to be determined is measured by referenceto the calibration curve established with IT-T101 introduced atdifferent known concentrations.

This test--by virtue of its principle of recognition--makes it possibleto measure the intact immunotoxin molecules.

Furthermore, comparison of the concentrations obtained in this assaywith those measured by the test for in vitro cytotoxic activity ontarget cells gives identical values, thereby ensuring that theimmunotoxin determined by the RIM-1 test corresponds to molecules whichhave retained their property of cytotoxicity.

(b) Measurement of the elimination kinetics of the antibody directedagainst human T cells (or antibody T101)

This antibody was prepared and purified in the manner indicated inFrench Patent Application No. 8121836. The antibody T101 is injectedintravenously into rabbits at a dose of 0.835 mg/kg. The plasma samplesare taken as previously. The antibody concentration in the samples ismeasured by radioimmunometric assay (RIM-2). This assay is performedunder the same conditions as the RIM-1 test, except that here the Ac1solution is a solution containing 10 mg/ml of goat antibodies directedagainst mouse IgG, purified by affinity chromatography. The antibody Ac2is identical to that in the RIM-1 test. The concentration of antibodyT101 in the samples to be determined is measured by reference to thecalibration curve established with the antibody T101 introduced atdifferent known concentrations.

(c) Measurement of the elimination kinetics of the A chain of ricin

The A chain of ricin was prepared and purified in the manner indicatedin French Patent No. 78/27838 and its Addition no. 79/24655 of the priorart. The A chain is injected intravenously into rabbits at a dose of0.415 mg/kg. The plasma samples are taken as previously. Theconcentration of A chain in the samples is measured by radioimmunometricassay (RIM-3). This assay is performed under the same conditions as theRIM-1 test, the antibodies Ac1, absorbed on the solid phase, also beingsheep antibodies directed against the A chain of ricin, purified byaffinity chromatography, and the antibodies Ac2 being the sameradiolabeled antibodies as described in RIM-1. The concentration of Achain of ricin in the samples to be determined is measured by referenceto a calibration curve established with the A chain of ricin introducedat different known concentrations.

The values of the concentrations of immunotoxin, antibody and A chain ofricin in the plasma, measured by these three tests, are reproducible,reliable and quantitative. The detection threshold for these threeproducts is 1 ng/ml. A study of the reproducibility within an assay andbetween assays gives variation coefficients below 10% for concentrationvalues within the range from 1 to 200 ng/ml.

B--Results:

The results of the experiments carried out are represented in the formof curves showing, on the abscissa, the time expressed in hours and, ona logarithmic scale on the ordinate, the concentration of the productmeasured, expressed as a percentage of the theoretical plasmaconcentration at time zero. This value, called the "relative plasmaconcentration" (RPC), is calculated using the following expression:##EQU1## The plasma volume is considered to be equal to 36 ml/kg of bodyweight of the animal.

(a) In the absence of mannane: plasma elimination kinetics of theimmunotoxin IT-T101, the antibody T101 and the A chain of ricin.

FIG. 1 shows the plasma elimination curve of IT-T101, which has twophases (curve 1). In the first phase, the product disappears rapidly(about 97% in 30 minutes); in the second phase, the decrease is slower.The first elimination phase observed with IT-T101 does not appear in theelimination kinetics of the antibody T101, where only one, slowelimination phase is recorded (curve 2). On the other hand, the plasmaelimination kinetics of the uncoupled A chain are very comparable tothose of IT-T101: 1 hour after injection, only 0.7% of the administereddose still remains in the plasma (curve 3).

(b) In the presence of mannane: plasma elimination kinetics of theimmunotoxin IT-T101 and the A chain of ricin.

The mannane was administered according to the following scheme:

20% of the final dose of the polysacchardie is injected intravenously 10minutes before the injection of IT-T101 or A chain. At time zero, 40% ofthe final dose of the polysaccharide is injected intravenously inassociation with IT-T101 or A chain (0.415 mg of A chain/kg). Then, 20%of the final dose of polysaccharide is injected intravenously at times1.5 hours and 5 hours respectively.

FIG. 2 shows the plasma elimination curve, as a function of time, ofIT-T101 injected intravenously in association with mannane at a totaldose of 0.416 g/kg (curve 2). In the presence of mannane, the firstelimination phase--responsible for the disappearance of the greater partof the product--is practically suppressed, leading to a major increasein the level of active immunotoxin in the plasma. 15 hours afterinjection, the concentration of IT-T101 is 100 times greater when theimmunotoxin has been associated with mannane than when mannane is absent(curve 1).

This effect of inhibiting the elimination of the immunotoxin from theplasma depends on the dose of mannane. Lower doses of mannane (166 mg/kgand 16.6 mg/kg) produce weaker effects (curves 3 and 4).

The properties of mannane are also observed with the uncoupled A chain,as shown in FIG. 3. This point confirms that the rapid disappearance ofthe immunotoxin is indeed attributable to the constituent A chain, inparticular because of its terminal mannose residues.

It is seen nevertheless that the first elimination phase of the A chainis not totally suppressed, contrary to what happens with theimmunotoxin. This confirms the totally surprising effect observed whenmannane and immunotoxin are administered simultaneously.

EXAMPLE 2

To show a possible specificity of action of mannane, the plasmaelimination kinetics of the immunotoxin IT-T101 were measured in thepresence of other polysaccharides not possessing mannose residues in theterminal position, these polysaccharides being the dextrans T10, T40 orT500 (glucose polymers with respective molecular weights of about10,000, 40,000 and 500,000) administered at a total dose of 416 mg/kg,galactan (galactose polymer) administered at a total dose of 166 mg/kg,and asialofetuine (highly glycosylated glycoprotein with terminalgalactose) administered at a total dose of 166 mg/kg.

The curves illustrated in FIG. 4 show that these polysaccharides havevirtually no effect on the plasma elimination kinetics of theimmunotoxin IT-T101.

EXAMPLE 3

This example demonstrates the hepatic capture of the A chain of ricinafter intravenous injection, and the inhibition of this capture bymannane.

The A chain radiolabeled with iodine 125 is injected intravenously intoCharles River France CD1 mice in the absence or presence of mannane at adose of 1 g/kg. At different times during the experiment, two animalsare anesthetized. The abdominal cavity is opened, the vena cava is cutand the liver is washed with 10 ml of physiological salt solution byinjection into the portal vein. The liver is totally removed and theradioactivity is determined. The results are represented as a percentageof the number of cpm fixed to the liver, relative to the total number ofcpm injected (FIG. 5). In the absence of mannane, the A chain of ricinis captured very quickly and efficiently by the liver, as indicated bythe radioactivity peak. Conversely, in the presence of mannane, thisradioactivity peak is practically suppressed. This result confirms thatthe A chain of ricin is trapped by the liver and that the maintenance ofthe immunotoxin, like the A chain, at high plasma levels in the presenceof mannane is indeed due to the inhibition of this hepatic capture.

EXAMPLE 4

This example demonstrates the absence of an antagonistic effect ofmannane towards the selective cytotoxicity of the immunotoxin IT-T101 invitro.

In these experiments, the cytotoxicity was evaluated by measuring theincorporation of ¹⁴ C-leucine by the target cells (CEM cells) afterincubation for 24 hours at 38° C. in the presence of knownconcentrations of the immunotoxin studied, or of reference cytotoxicsubstances, in the absence or presence of mannane at a concentration of10 mg/ml. The technique employed is the one described previously (J.Biol. Chem. 1984, 259 (15), 9359).

A check was carried out beforehand to show that mannane is not cytotoxicto the cells at the concentrations used. The results of theseexperiments are shown in Table I. The cytotoxic effect is measured bythe value of the molar concentration (IC₅₀), expressed as A chain, whichcauses a 50% inhibition of the incorporation of the tracer.

The immunotoxin, by itself or in its form activated by ammoniumchloride, fully retains its activity. In the same way, the intrinsictoxicity of the A chain is not modified. Thus, in the presence ofmannane, the characteristic cytotoxic properties of the immunotoxin arenot affected.

                  TABLE I                                                         ______________________________________                                                      IC.sub.50 expressed as molarity of                                            A chain                                                         Substances                    with mannane                                    tested          without mannane                                                                             (10 mg/ml)                                      ______________________________________                                        Immunotoxin IT-T101                                                                           3.0 · 10.sup.-13 M                                                                 2.8 · 10.sup.-13 M                     plus NH.sub.4 Cl                                                              IT-T101         1.0 · 10.sup.-9 M                                                                  1.2 · 10.sup.-9 M                      A chain         7.0 · 10.sup.-7 M                                                                  7.0 · 10.sup.-7 M                      ______________________________________                                    

EXAMPLE 5

Toxicity of the immunotoxin IT-T101 injected into mice in associationwith mannane.

It was important to check the overall toxicological effect of theassociation of immunotoxin plus mannane on the whole animal. This wasdone by determining the 50% lethal dose of the antimelanoma immunotoxin(IT-HM) administered intravenously to Charles River France CD1 mice inthe absence of mannane or with the intravenous co-administration of 10mg of mannane per mouse. The preparation and cytotoxic properties ofthis antimelanoma conjugate (IT-HM) were described in French PatentApplication No. 81/07596.

The values found are indicated in Table II.

                  TABLE II                                                        ______________________________________                                        Product tested LD.sub.50                                                      ______________________________________                                        IT-HM by itself                                                                              460 micrograms/mouse                                           IT-HM + 10 mg of                                                                             115 micrograms/mouse                                           mannane/mouse                                                                 ______________________________________                                    

These results show a slight increase in toxicity of the immunotoxin whenit is administered simultaneously with mannane. This increase intoxicity by a factor of only 4 does not restrict the in vivo use ofmannane in view of the very significant effects in respect ofmaintaining the plasma concentration of immunotoxins in vivo, asdemonstrated above.

EXAMPLE 6

The purpose of this example is to demonstrate the effect of mannane onthe action of an immunotoxin in an "in vivo" experiment.

The experiment was carried out on BL 1.1 mice (negative Thy 1.2 cells)[International Journal of Cancer 24, 168-177, (1979)]. The immunotoxinused is the conjugate in which the antibody directed against Thy 1.2(antibody AT15E) is associated by means of a disulfide bond with the Achain of ricin, and which is prepared by the process described in ourApplications of the prior art.

The following protocol was used.

On day 0, groups of 10 BL 1.1 mice receive 5×10⁴ T2 cells (positive Thy1.2 cells of murine lymphoma) by intravenous injection and arerandomized before treatment.

The treatment is carried out intravenously on day 1:

1 group receives 10 μg/mouse of the conjugate antibody AT15E/A chain ofricin by itself;

1 other group receives the same quantity of the same conjugate mixedwith 10 mg of mannane.

In addition, 4 control groups respectively receive:

the culture medium RPMI (medium used for the culture of the T2 cells);

mannane by itself (10 mg/mouse);

the A chain of ricin (10 μg) and mannane (10 mg);

the A chain of ricin (10 μg), the antibody AT15E (30 μg) and mannane (10mg).

The animals are observed for 50 days and the mortality is noted.

FIG. 6 shows the percentage of animals surviving as a function of thetime which has elapsed since the treatment.

Curve 1 relates to the animals which received the immunotoxin by itselfand curve 2 relates to the animals which received the combination ofimmunotoxin plus mannane.

As can be seen, the immunotoxin/mannane association gave a survival rateof 90% fifty days after the treatment, whereas the administration of theimmunotoxin by itself gave an animal survival rate of only 30% after 50days.

Furthermore, the following observations were made on day 50 for thecontrol groups (not shown on FIG. 6):

0% survival rate for the animals treated with RPMI and with A chain plusmannane;

10% survival rate for the animals receiving mannane by itself;

20% survival rate for the animals treated with A chain ofricin+antibody+mannane.

These results show the efficacy of the immunotoxin/mannane combinationcompared with the immunotoxin used by itself and compared with thecontrol substances.

The combination consisting of an immunotoxin and mannane can thereforebe used as a drug in human therapy. This combination can be used for thetreatment of cancerous or non-cancerous complaints in which the targetcells will be recognized by the antibody used to prepare theimmunotoxin.

With the aim of eliminating all the target cells, the treatment must becarried out with a sufficient dose of immunotoxin associated with aquantity of mannane which can vary from 10 mg to 1 g/kg for eachadministration of immunotoxin. The optimum modalities for administrationof the constituents of the combination, and also the duration of thetreatment, will have to be determined in each case according to thesubject and the nature of the complaint to be treated.

The new drugs accordingg to the invention are packaged foradministration by injection, preferably intravenous injection. Theconstituents of the combination will preferably be stored separately andmay be mixed as required, for immediate use, in the syringe or theperfusion solvent.

What is claimed is:
 1. A pharmaceutical composition comprising acombination of at least one immunotoxin and at least onemannose-containing polymer.
 2. A pharmaceutical composition as claimedin claim 1, wherein the immunotoxin comprises a natural, semi-syntheticor synthetic toxin, toxic sub-unit or fragment of a toxic sub-unitcomprising polysaccharide groups containing mannose residues and anantibody component; and the mannose-containing polymer is a polyoside orpolysaccharide carbohydrate polymer which has an average molecularweight greater than 1000 and contains a large portion of mannoseresidues.
 3. A pharmaceutical composition as claimed in claim 1 whereinthe immunotoxin is anti-T65 coupled to the A-chain of ricin and themannose-containing polymer is a mannan.
 4. A pharmaceutical compositionaccording to claim 2 packaged for administration by injection.
 5. Apharmaceutical composition as claimed in claim 2 wherein themannose-containing polymer contains from 20-100% of mannose residues. 6.A method to inhibit the rapid plasma elimination of immunotoxinscomprising a natural, semisynthetic or synthetic toxin, toxic sub-unitor fragment of a toxic sub-unit comprising polysaccharide groupscontaining mannose residues joined to an antibody component whichcomprises providing an effective amount for inhibiting the eliminationof the immunotoxin from the plasma of at least one mannose-containingpolymer.
 7. A method as claimed in claim 6 wherein themannose-containing polymer is mannan.
 8. A method as claimed in claim 6wherein the effective amount of at least one mannose-containing polymeris from 10 mg to 1 g/kg for each administration of immunotoxin.
 9. A kitfor the preparation of a pharmaceutical composition comprising acombination of at least one immunotoxin and at least onemannose-containing polymer, and wherein the immunotoxin and themannose-containing polymer are packaged separately within the kit and atleast a portion of the immunotoxin and the mannose-containing polymerare mixed prior to use.
 10. A kit as claimed in claim 9 wherein theimmunotoxin is anti-T65 coupled to the A chain of ricin and themannose-containing polymer is mannan.